Field of the Invention
The present invention relates to an enzymatic immunoassay by which a minute amount of biological substances can be detected and/or estimated.
As one of known immunological methods to detect and/or estimate a minute amount of biological substances, the enzymatic immunoassay is recently recognized as noteworthy and actively developed through investigations, in which an enzyme is used as a label on an antigen-antibody reaction complex. This method is an alternative to the hitherto used radio-immunoassay which is carried out using radioisotopes and requires much cares in handling.
The enzymatic immunoassay may be realized in some different manners, as taught for example in "Clinical Chemistry", Vol. 22, No. 8, 1243-1255(1976). However, in general, the procedure goes as follows. At first an antigen or an antibody is affixed to the surface of the inner wall of a cell as reaction chamber or on the surface of beads placed in the cell, the antigen or antibody is brought into an antigen-antibody reaction with a conjugate which is combined with an enzyme as label to form a complex, then a substrate is introduced in the cell which, by the activity of the enzyme, gives rise to an optically detectable change (such as in fluorescent intensity, absorbance, and emission intensity), the change is measured by an optical means such as a fluoro-photometer, and an absorbance photometer, and from the result of measurement the amount of the complex containing enzyme, or hence the amount of the antigen or antibody.
In the enzymatic immunoassay which is concerned with the determination of an amount as minute as 10.sup.-13 of biological substances in general, a relatively large error is involved in the estimation and therefore a high performance of the instrument for measurements as well as removal of error sources are ardently required.
With these points in mind, the present inventors investigated the conventional processes in general of enzymatic immunoassay and found the problems described below.
The processes in the enzymatic immunoassay are generally carried out as follows: at first, an antigen-antibody-enzyme complex is formed affixed to the inner wall of the cup as the reaction chamber, on the surface of beads placed in the cup, or on an insoluble carrier, and then the complex is brought into contact with a substrate solution to start the enzyme reaction. The enzymatic immunoassay is conducted by measuring the fluorescence intensity of the substrate after a certain period of time, either stopping the enzyme reaction with a stopping solution or not. However, in these processes, the time from the initiation of enzyme reaction to the measurement should be strictly controlled, and so-called zero point correction is needed for the fluorescence intensity of the substrate because the intensity at the starting point is not always zero but shows some dispersion. Further, in the characteristic curve expressing the increase of the fluorescence intensity at the substrate, the rate of increase in the intensity becomes gradually smaller from the initiation of the enzyme reaction, and in addition the characteristic curve varies depending on the difference in the amount of enzyme. The correlation between two curves is not linear and hence errors due to difference in the measuring time are different in magnitude and depend individually on the characteristic curves.